EFFECT OF HEMOGLOBIN MEMBRANE INTERACTIONS ON RED CELL FUNCTION

这个项目的目的是测试假设， 具有特定膜成分的突变和未配对血红蛋白链 影响KCl协同转运和红细胞（RBC）的变形性。 两 具体目标如下：（1）确定 突变的未配对Hb链改变KCl共转运、膜变形性 和机械强度，以及2）识别 与Hb相互作用以介导这些作用。 定义（IOV）并重新密封 在正常Hb与突变Hb和过量α存在下的膜， β珠蛋白链 差示提取和再添加选定的 膜骨架组件将通过以下方式测试药物对Hb的影响： 膜骨架，以及阻止Hb链结合的策略 与完整的膜蛋白将测试药物的B3和 糖蛋白 不同类型血红蛋白对细胞膜的不同影响 变形性和机械强度将通过比较 含有各种浓度和类型的再密封膜的测定 血红蛋白，使用ektacytometer，衍射粘度计。 以识别 与Hb相互作用以调节这些的特定膜组分 功能，RBC将用各种交联剂处理， 低浓度以稳定完整的Hb/膜结合 细胞，并防止其在分析过程中被破坏。 Hb交联 通过分离膜检测特定的膜组分 蛋白质通过聚丙烯酰胺凝胶电泳，制备免疫印迹， 用抗血红蛋白和抗膜成分的抗体探测它们。 将确认Hb和膜组分之间的表观复合物 通过用抗膜组分的抗体进行免疫沉淀，和 使用抗Hb探测免疫沉淀物的免疫印迹。 探讨 对于Hb与介导KCl共转运的蛋白质的相互作用， 将制备针对该蛋白的兔多克隆抗体， 利用抗原在高钾和低钾条件下的差异表达， 绵羊红细胞 预计从这些研究中获得的信息 将提供关于HbS和HbC对膜的影响的新信息 可能有助于理解变量的函数 镰状病的病理生理表现。

The objective of this project is to test the hypothesis that interaction of mutant and unpaired Hb chains with specific membrane components affects KCI cotransport and deformability of red cells (RBC). Two Specific Aims will be pursued: 1) to determine the extent to which mutant unpaired Hb chains modify KCI co-transport, membrane deformability and mechanical strength, and 2) to identify membrane components that interact with Hb to mediate these effects. To define (IOV) and resealed membranes in the presence of normal Hb vs mutant Hb and excess alpha and beta globin chains. Differential extraction and readdition of selected membrane skeletal components will test for medication of Hb effects via the membrane skeleton, and maneuvers to block association of Hb chains with integral membrane proteins will test for medication by B3 and glycophrin. Differential effects of various type of Hb on membrane deformability and mechanical strength will be assessed by comparative assays of resealed membranes containing various concentrations and types of Hb, using the ektacytometer, a diffraction viscometer. To identify specific membrane components that interact with Hb to modulate these functions, RBC will be treated with variety of cross-linking agents at low concentrations to stabilize Hb/membrane associations in the intact cell and prevent their disruption during analysis. Hb cross-linking to specific membrane components will be detected by separating membrane proteins by polyacrylamide gel electrophoresis, preparing immunoblots and probing them with anti-Hb and antibodies against membrane components. Apparent complexes between Hb and membrane components will be confirmed by immunoprecipitation with antibody against the membrane component, and probing of immunoblots of the immunoprecipitate using anti-Hb. To probe for interaction of Hb with the protein that mediates KCI cotransport, rabbit polyclonal antibodies against the protein will be prepared, making use of the differential expression of the antigen in high K and low K sheep RBC. It is expected that information derived from these studies will provide new information about the effects of HbS and HbC on membrane function that may be useful in understanding the variable pathophysiologic manifestations in the sickling disorders.